During translation the genetic code is interpreted into polypeptide cell components. Mistakes at this level can have deleterious effects on cell viability by altering protein function and stability. Aminoacyl-tRNA synthetases (AARSs) are directly involved in translational fidelity. They couple amino acids to tRNA molecules bearing corresponding anti-codon sequences. Several AARSs have evolved interesting proofreading mechanism to maintain high substrate specificity in vivo. Specifically, a handful-possess a hydrolytic editing activity to correct errors initially made at their synthetic sites. The editing and synthetic activities of AARSs occur at distally located active sites. Therefore, misacylated substrates must "translocate" between catalytic centers. Substrate translocation has been demonstrated essential, but is poorly understood. This proposal describes experiments that will elucidate events surrounding the process. First, an in vivo saturation mutagenesis screen will be used to identify components of the translocation machinery. The components will then be characterized biochemically to determine their role in substrate translocation. The goal is to produce a refined model for substrate translocation and AARS catalyzed editing.